CN114480323B

CN114480323B - Oat glycosyltransferase AsUGT73E1 and application thereof in steroid saponin synthesis

Info

Publication number: CN114480323B
Application number: CN202011251664.7A
Authority: CN
Inventors: 薛哲勇; 宋伟; 苏同兵
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2023-10-13
Anticipated expiration: 2040-11-11
Also published as: CN114480323A

Abstract

The invention relates to a saponin metabolic pathway, in particular to oat glycosyltransferase AsUGT73E1 and application thereof in steroid saponin synthesis. The glycosyltransferase provided by the invention is protein as described in the following a1 or a 2: a1. a protein with an amino acid sequence shown as SEQ ID NO. 1; a2. and (3) a protein with glycosyltransferase activity, which is formed by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1. The glycosyltransferase can take the trilobatin or the pennogenin-3-O-glucoside as a substrate, introduce rhamnosyl, and generate the paris polyphylla saponin V or the paris polyphylla saponin VI.

Description

Oat glycosyltransferase AsUGT73E1 and application thereof in steroid saponin synthesis

Technical Field

The invention relates to a saponin metabolic pathway, in particular to oat glycosyltransferase AsUGT73E1 and application thereof in steroid saponin synthesis.

Background

The paris polyphylla is a general term of plants in paris of the family lily, can be used as a main raw material of Chinese patent medicines such as Yunnan white drug powder, gongxuening, heat toxin clearing and the like, and has extremely high medicinal and economic values. Steroid saponins are the main chemical components of Paris polyphylla plants, 160 species have been identified at present, and mainly comprise Paris polyphylla saponins (polyphelin) I, II, III, V (dioscin) and VI, VII (pennogenin), and the pharmacological activity is wide. Antitumor is the main effect of paris saponin, paris saponin I induces autophagy and cell cycle arrest by inhibiting PDK1/Akt/mTOR signaling pathway in human gastric cancer HGC-27 cells and down regulating cyclin B1 (He et al 2019). Paris polyphylla VI induces apoptosis and autophagy in non-small cell lung cancer via ROS-triggered mTOR signaling pathway (Teng et al 2019). Paris polyphylla saponin VII can promote mitochondria to generate ROS and activate MAPK and PTEN/p53 pathways, and jointly induce apoptosis of HepG2 human liver cancer cells (Zhang et al 2016). The paris polyphylla saponin VII has good effect in resisting bacteria and diminishing inflammation, can remarkably inhibit the growth of dendritic cladosporium, candida and propionibacterium acnes, and can be used as an effective substitute of synthetic medicines (Deng et al 2008The method comprises the steps of carrying out a first treatment on the surface of the Qin et al 2012). Diosgenin (diosgenin) can enhance activities of lipoprotein lipase, liver lipase, superoxide dismutase, glutathione peroxidase and nitric oxide synthase of hyperlipidemic mice, improve lipid distribution, and have effect of reducing blood lipid (Gong et al 2010). Paris polyphylla saponin III has excellent insect repellent activity, and can kill dactylogyrus (EC) parasitizing in gill part of goldfish ₅₀ ＝18.06mg l ^-1 ) And is low toxic to goldfish (Wang et al 2010). In the new coronapneumonia study, the saponin molecules were found to have potential anti-new coronavirus activity. By docking screening, it is speculated that paris saponin I binds to 2019-nCoV major protease (M protease) and prevents viral replication (Yan et al 2020).

The research of paris polyphylla saponin at the present stage mainly focuses on medicine and clinic, so that the understanding of metabolic pathways, especially downstream biosynthesis processes, is quite lacking. 2,3-oxidosqualene (2, 3-oxadiquatene) is a common precursor for sterol and triterpene synthesis, and a sterols or triterpene skeleton is produced under the catalysis of 2,3-oxidosqualene cyclase (2, 3-Oxidosqualene cyclases, OSCs). Most pentacyclic triterpene synthases are capable of catalyzing the formation of a dammarane type cation in the "hair-hair" conformation from 2, 3-oxidized squalene, followed by further rearrangement to yield pentacyclic triterpenes such as α -amyrin, β -amyrin, lupeol (lupeol) (Xue et al 2018). Cycloartenol synthase (cycloartenol synthase) catalyzes the formation of a presteroid cation in a "pair-coat-pair" conformation from 2, 3-oxidized squalene, followed by conversion to cycloartenol, and synthesis of cholesterol through a series of reactions. The cholesterol content in plants, although generally low, is an important component of plant sterols (phytosterol) and is also a direct precursor of steroidal saponins (C. Rdenas et al 2015). The synthesis of steroidal sapogenins requires hydroxylation of cholesterol side chains, and is mainly modified by cytochrome P450 enzymes (CYPs). For example, CYP90Bs are found in Paris polyphylla (Paris polyphella) to evolve sterol polyhydroxyenzyme activity by gene replication, ppCYP90G4 can catalyze hydroxylation of cholesterol at C16 and C22 with E ring closure. 16,22 (S) -dihydroxycholesterol is further hydroxylated at position C26 to form diosgenin (Christ et al 2019) under the action of PpCYP94D108 and other enzymes, while pennogenin (pennogenin) at position C17P 450 enzyme remains to be resolved. Finally, diosgenin or pennogenin is glycosylated under the action of glycosyltransferase (UDP-glycosyltransferase, UGT) to form rhizoma paridis saponin with various biological activities. Currently, there are few reports on the study of glycosylation modification of steroidal sapogenins.

The paris polyphylla saponin is basically derived from plant extraction, however, due to excessive development, the resources of paris polyphylla are exhausted. The use of synthetic biology to construct heterologous biosynthetic pathways is becoming an effective way to obtain natural active ingredients. However, the paris plant has huge genome and numerous saponins, and it is difficult to analyze the metabolic pathway of paris saponins through the gene co-expression network. Oat (Avena sativa l.) is the only saponin-rich plant in the grasses (Vincken et al 2007) and two different types of saponins can be synthesized. One is the triterpene saponin-avenacins (avenacins) synthesized in the root and root tip, and the other is the steroid saponin-avenacins (avenacins) accumulated in leaves and grains. Oat is annual herb, has short growth cycle, can complete one growth cycle under long sunlight condition for 3-4 months, and can be used as an ideal mode material for researching the synthesis path of steroid saponin. With the discovery of the triterpene saponin avenacins metabolic gene cluster, the synthesis way of oat triterpene saponin has been completely analyzed through many years of research (Louveau et al 2018; orme et al 2019).

Disclosure of Invention

In order to analyze the metabolic pathway of the paris polyphylla saponin and realize the artificial synthesis of the paris polyphylla saponin, two oat glycosyltransferases which are respectively named as AsUGT73E5 and AsUGT73E1 are obtained by taking oat as a mode material. To verify glycosyltransferase function, we designed PCR primers for coding regions of glycosyltransferase genes AsUGT73E5 and AsUGT73E1, asUGT73E5-ORF-F/R, asUGT E1-ORF-F/R. PCR amplification is carried out by taking the oat seedling cDNA after reverse transcription as a template, and the AsUGT73E5 and AsUGT73E1 genes are obtained. The open reading frame of the AsUGT73E1 gene contains 1473 bases, the nucleotide sequence of the gene is shown as SEQ ID NO. 2, and the coded amino acid sequence is shown as SEQ ID NO. 1. The open reading frame of the AsUGT73E5 gene contains 1548 bases, the nucleotide sequence of the AsUGT73E5 gene is shown as SEQ ID NO. 4, and the coded amino acid sequence is shown as SEQ ID NO. 3.

Then, we performed protein expression and purification. The Open Reading Frames (ORFs) of the genes AsUGT73E5 and AsUGT73E1 are cloned into a prokaryotic expression vector pGEX-6p-1 by homologous recombination, and expression competent Rosetta (DE 3) is transformed. Cultivated in Amp-resistant LB liquid medium to od600=0.6, and induced overnight at low temperature with addition of IPTG. After the cells were sonicated, proteins in the supernatant were purified by Glutathione Beads, and each fraction was collected for SDS-PAGE analysis (FIG. 2). The recombinant proteins all appeared to have a distinct band around 80kDa compared to the uninduced control samples. The AsUGT73E5 and AsUGT73E1 proteins respectively comprise 515 and 490 amino acids, and the molecular weights of the fused AsUGT73E5 and AsUGT73E1 proteins are 82.21KDa and 79.79KDa respectively.

Next, we used diosgenin, pennogenin and neoacteoside to perform functional verification on glycosyltransferases AsUGT73E5 and AsUGT73E1, respectively, with the following results:

HPLC detection is carried out on the product of the reaction of AsUGT73E5 protein, diosgenin and UDP-Glc (figure 3), and compared with the no-load reaction, the product of AsUGT73E5 shows a new peak (product 1) at 22.3min, and the peak outlet time is the same as that of a trillin standard product. The sample was recovered, and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a new product peak (product 2) was observed at 20.7min, which time was consistent with the rhizoma paridis saponin V standard. TOF positive ion scanning mode detection is shown in FIG. 4, the molecular weights of product 1 and product 2 are 577.38 (M+H+) and 723.43 (M+H+), respectively, and are consistent with those of trilobatin (576.3) and parietal saporin V (722.4), which shows that parietal sapogenin is continuously catalyzed by AsUGT73E5 and AsUGT73E1 to generate parietal saporin V.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with pennogenin, UDP-Glc (FIG. 5), the product of AsUGT73E5 showed a new peak at 18.8min (product 3) compared with the empty reaction. The sample was recovered, and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a new product peak (product 4) was observed at 17.1min, which time was consistent with the Paris polyphylla saponin VI standard. TOF positive ion scanning mode detection is shown in FIG. 6, the molecular weights of product 3 and product 4 are 593.37 (M+H+) and 739.43 (M+H+), respectively, and are consistent with the molecular weights of pennogenin-3-O-glucoside (592.3) and parisonin VI (738.4), which indicates that pennogenin is continuously catalyzed by AsUGT73E5 and AsUGT73E1 to generate parisonin VI.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with neoacteosin, UDP-Glc (FIG. 8), the product of AsUGT73E5 showed a new peak at 16.7min (product 5) compared to the empty reaction. The sample was recovered and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a second product peak (product 6) was present at 15.6 min. TOF positive ion scanning mode detection is shown in FIG. 9, the molecular weights of product 5 and product 6 are 593.37 (M+H) ⁺ ) And 739.43 (M+H) ⁺ ) Indicating that the neotame sapogenin generates corresponding glycosylation products after being continuously catalyzed by AsUGT73E5 and AsUGT73E1.

Based on the above studies, the present invention provides a glycosyltransferase which is a protein as described in a1 or a2 below:

a1. a protein with an amino acid sequence shown as SEQ ID NO. 1;

a2. and (3) a protein with glycosyltransferase activity, which is formed by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

Genes encoding the glycosyltransferases are also within the scope of the invention.

In some embodiments of the invention, the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

Expression cassettes, vectors or recombinant bacteria containing said genes are also within the scope of the invention.

In some embodiments, the vector is a cloning vector comprising the gene encoding the glycosyltransferase and elements required for plasmid replication, e.g., pClone007 Blunt Simple Vector into which the encoding gene is inserted. In other embodiments, the vector is an expression vector comprising a gene encoding the glycosyltransferase and an element capable of successful protein expression, e.g., pGEX-6p-1 vector into which the encoding gene is inserted.

In some embodiments, the recombinant bacterium is a recombinant bacterium comprising a cloning vector, such as e.coli DH5 a, and the gene encoding the glycosyltransferase is replicated by culturing the recombinant bacterium. In other embodiments, the recombinant bacterium is a recombinant bacterium comprising an expression vector, and the recombinant bacterium is cultured under suitable conditions, e.g., with the addition of an appropriate amount of IPTG, at 16 ℃ to induce expression of the glycosyltransferase.

The invention also provides a preparation method of the glycosyltransferase, which comprises the following steps: constructing an expression vector of the coding gene of the glycosyltransferase, introducing the expression vector into expression host bacteria to obtain recombinant bacteria, culturing the recombinant bacteria and inducing protein expression, collecting thalli, and extracting and purifying protein.

The use of the glycosyltransferase in a glycosyl transfer reaction is also within the scope of the present invention.

The use of the glycosyltransferase in the synthesis of steroid saponins also falls within the scope of the present invention. The steroid saponin synthesis includes in vitro synthesis and in vivo synthesis, for example, synthesis of steroid saponins in microorganisms (e.g., yeast) or plants.

In some embodiments of the invention, in the synthesis of the steroid saponin, the substrate is trillion saponin or pennogenin-3-O-glucoside, and rhamnosyl is introduced under the action of glycosyltransferase to generate paris saponin V or paris saponin VI.

The invention also provides a method for synthesizing paris polyphylla saponin V, which comprises the following steps: and (3) reacting the glycosyltransferase with the trillin and UDP-Rha to generate the paris saponin V.

The invention also provides a method for synthesizing paris polyphylla saponin VI, which comprises the following steps: the glycosyltransferase reacts with the pennogenin-3-O-glucoside and UDP-Rha to generate the paris polyphylla saponin VI.

The glycosyltransferase provided by the invention is helpful for exploring the synthetic way of plant steroid saponins, and simultaneously provides gene resources for obtaining a large amount of paris polyphylla saponins or other saponins.

Drawings

FIG. 1 shows the electrophoresis patterns of amplification products of AsUGT73E5 and AsUGT73E1 genes. M is DL 2000;1 is AsUGT73E5 gene band; 2 is AsUGT73E1 gene band.

FIG. 2 shows SDS-PAGE patterns of prokaryotic expression of AsUGT73E5 and AsUGT73E1 proteins. M: a Page-roller protein molecular marker; 1: not induced; 2: whole cell proteins after IPTG induction; 3: cell supernatant after IPTG induction; 4: cell precipitation after IPTG induction; 5: purifying the supernatant GST column; 6: and (5) purifying protein, and concentrating by ultrafiltration.

FIG. 3 chromatogram of glycosylation products of AsUGT73E5, asUGT73E1 reaction with diosgenin. The retention time (min) is on the abscissa and the electrical signal (mAU) is on the ordinate. pGEX-empty: protein extracted and purified from cells containing pGEX-6P-1 empty vector after protein induction expression is used as a blank control; pGEX-AsUGT73E5: the cells containing pGEX-AsUGT73E5 vector are subjected to protein induction expression, and then extracted and purified to obtain AsUGT73E5 protein; cells containing pGEX-AsUGT73E1 vector are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 4 mass spectrometry analysis of glycosylation products of AsUGT73E5, asUGT73E1 reactions with diosgenin. The abscissa is mass-to-charge ratio and the ordinate is ionic strength.

FIG. 5A chromatogram of the glycosylation products of AsUGT73E5, asUGT73E1 reactions with pennogenins. The retention time (min) is on the abscissa and the electrical signal (mAU) is on the ordinate. pGEX-empty: protein extracted and purified from cells containing pGEX-6P-1 empty vector after protein induction expression is used as a blank control; pGEX-AsUGT73E5: the cells containing pGEX-AsUGT73E5 vector are subjected to protein induction expression, and then extracted and purified to obtain AsUGT73E5 protein; cells containing pGEX-AsUGT73E1 vector are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 6 mass spectrometry analysis of glycosylation products of AsUGT73E5, asUGT73E1 reactions with pennogenins. The abscissa is mass-to-charge ratio and the ordinate is ionic strength.

FIG. 7.AsUGT73E5 and AsUGT73E1 catalyze the process of steroidal sapogenin glycosylation.

FIG. 8A chromatogram of the glycosylation products of AsUGT73E5, asUGT73E1 reactions with neoacteoside. The retention time (min) is on the abscissa and the electrical signal (mAU) is on the ordinate. pGEX-empty: protein extracted and purified from cells containing pGEX-6P-1 empty vector after protein induction expression is used as a blank control; pGEX-AsUGT73E5: the cells containing pGEX-AsUGT73E5 vector are subjected to protein induction expression, and then extracted and purified to obtain AsUGT73E5 protein; cells containing pGEX-AsUGT73E1 vector are subjected to protein induction expression, and then extracted and purified AsUGT73E1 protein.

FIG. 9 mass spectrometry analysis of glycosylation products of AsUGT73E5, asUGT73E1 reactions with neoacteoside. The abscissa is mass-to-charge ratio and the ordinate is ionic strength.

Detailed Description

The invention is further described below in connection with the following examples, which are to be understood as merely illustrative and explanatory of the invention, and are not in any way limiting of the scope of the invention.

Experimental materials:

the plant material used in the following experiments was diploid oat (Avena strigosa, S75), a known variety described in non-patent document Papadopoulou et al 1999. The above-mentioned biological materials are also stored in the laboratory, and the applicant states that they can be issued to the public for verification experiments within twenty years from the date of application.

Coli (Escherichia coli) DH 5. Alpha. And Rosetta (DE 3) were purchased from Shanghai Biotechnology Inc. Cloning vector pClone007 Blunt Simple Vector was purchased from the biotechnology company of new industry, beijing, family. Prokaryotic expression vector pGEX-6P-1 is stored in the laboratory and is also commercially available.

PCR primer:

the main reagent comprises:

diosgenin: CAS number: 512-04-9, molecular formula: c (C) ₂₇ H ₄₂ O ₃ The english name diongenin is purchased from the adult tuo pren technology development limited under the trade designation BP0504.

Pennogenin: CAS number: 507-89-1, molecular formula: c (C) ₂₇ H ₄₂ O ₄ English name pennogin. In experiments makeThe pennogenin is obtained by enzymatic separation and purification of paris polyphylla saponin VI, reference being made to Li, w, wang, z, gu, j, chen, l, hou, w, jin, y.p.,&wang, Y.P. (2015) Bioconversion of ginsenoside Rd to ginsenoside M1 by snailase hydrolysis and its enhancement effect on insulin secretion in v.die Pharmazie,70:340-346. Pennogenin is also commercially available.

Neoacteoside aglycone: CAS number: 6811-35-4, molecular formula: c (C) ₂₇ H ₄₂ O ₄ English name nuatigenin. The neotame sapogenin used in the experiments was obtained by enzymatic separation and purification of an oat saponin (avenacides) extract, and the extraction method of oat saponins (avenacides) was described in reference to Yang, j.l., wang, p., wu, w.b., zhao, y.t., idehen, e.,&Sang,S.M.(2016).Steroidal saponins in oat bran.Journal of Agricultural&food Chemistry, 64 (7): 1549-1556, the enzymatic separation and purification method is described in reference to Li, w, wang, z, gu, j, chen, l, hou, w, jin, y.p.,&Wang,Y.P.(2015).Bioconversion of ginsenoside Rd to ginsenoside M1 by snailase hydrolysis and its enhancement effect on insulin secretion in vitro.Die Pharmazie,70:340–346.。

UDP-Glucose (UDP-Glc): CAS number: 28053-08-9, molecular formula: c (C) ₁₅ H ₂₂ N ₂ Na ₂ O ₁₇ P ₂ Purchased from Beijing Cool Bo technologies Inc., cat# CU11611-500mg.

UDP-Rhamnose (UDP-Rha): CAS number: 1526988-33-9, molecular formula: c (C) ₁₅ H ₂₂ N ₂ Na ₂ O ₁₆ P ₂ The English name UDP 5' -biphospho-a-L-rhamnose, purchased from Souzhou Hanse Biotechnology Co.

Trillin: CAS number: 14144-06-0, molecular formula: c (C) ₃₃ H ₅₂ O ₈ Purchased from chengdou pren technology development limited under the trade designation BP1124.

Paris polyphylla saponin V: CAS number: 19057-67-1, molecular formula: c (C) ₃₉ H ₆₂ O ₁₂ Purchased from chengdoupren technology development limited under the trade designation BP1151.

Paris saponin VI: CAS number: 55916-51-3, molecular formula: c (C) ₃₉ H ₆₂ O ₁₃ Purchased from chengdoupren technology development limited, cat No. BP1131.

Chromatographic methanol: purchased from merck, usa, cat No. 1.06007.4008.

Unless otherwise specified, the reagents used in the examples below are all conventional in the art and are commercially available or formulated according to conventional methods in the art; the experimental methods and conditions used are all those conventional in the art, and reference may be made to the relevant experimental manuals, well-known literature or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 discovery, cloning and expression of glycosyltransferase genes AsUGT73E5 and AsUGT73E1

1. Gene discovery

We have discovered two glycosyltransferase genes, designated AsUGT73E5 and AsUGT73E1, using oat as a model material. The Open Reading Frame (ORF) of the AsUGT73E1 gene contains 1473 bases, the nucleotide sequence of which is shown as SEQ ID NO. 2, and the coded amino acid sequence of which is shown as SEQ ID NO. 1. The Open Reading Frame (ORF) of the AsUGT73E5 gene contains 1548 bases, the nucleotide sequence of which is shown as SEQ ID NO. 4, and the coded amino acid sequence of which is shown as SEQ ID NO. 3. By transcriptome-target metabolite association analysis (Pearson correlation), we found that expression of these two glycosyltransferases was highly synergistic with cholesterol synthesis in oat 10 tissues and thus likely involved in steroid saponin synthesis. To verify the function of these two glycosyltransferases in steroid saponin synthesis, we performed gene cloning and expression.

2. Gene cloning

2.1 extraction of total RNA from oat seedlings

RNA was extracted using the method of Tiangen biosystems RNAprep Pure Plant Kit (product number: DP 441) and was performed according to the instructions for the kit, as follows:

(1) 50-100mg oat leaves are rapidly ground into powder in liquid nitrogen, 450 mu L of RL (beta-mercaptoethanol is added before use) is added, and vortex violent shaking and mixing are carried out;

(2) Transferring the solution to a filter column CS, centrifuging at 12,000rpm for 2-5min, and sucking the supernatant in the collecting tube into a centrifugal tube of RNase-Free;

(3) Adding absolute ethyl alcohol with the volume of 0.5 times of the supernatant, mixing uniformly, transferring the obtained solution and the precipitate into an adsorption column CR3, centrifuging at 12,000rpm for 30-60sec, pouring out waste liquid, and placing the adsorption column CR3 into a collecting pipe;

(4) Adding 350 μL deproteinized liquid RW1 into the adsorption column CR3, centrifuging at 12,000rpm for 30-60sec, pouring out waste liquid, and placing the adsorption column CR3 back into a collecting pipe;

(5) Preparing DNase I working solution: taking 10 mu L of DNase I storage solution, putting the DNase I storage solution into a new RNase-Free centrifuge tube, adding 70 mu L of RDD buffer solution, and gently mixing the two;

(6) Adding 80 mu L of DNase I working solution into the center of an adsorption column CR3, and standing at room temperature for 15min;

(7) 350. Mu.L deproteinized liquid RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm for 30-60sec, the waste liquid was discarded, and the adsorption column CR3 was returned to the collection tube;

(8) Adding 500 μl of rinsing liquid RW (ethanol is added before use) into the adsorption column CR3, standing at room temperature for 2min, centrifuging at 12,000rpm for 30-60sec, pouring out the waste liquid in the collection tube, and placing the adsorption column CR3 back into the collection tube, and repeating for one time;

(9) Centrifuging at 12,000rpm for 2min, pouring out the waste liquid, standing the adsorption column CR3 at room temperature for several minutes, and thoroughly airing the residual rinsing liquid;

(10) Placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and suspending and dripping 30-100 μl RNase-Free ddH into the middle part of the adsorption film ₂ O, left at room temperature for 2min, and centrifuged at 12,000rpm for 2min to obtain an RNA solution.

2.2 Synthesis of cDNA

SuperScript III Rreverse Transcriptase kit (Invitrogen, cat# 18080085) was used and operated as described in the kit instructions:

(1) RNA template denaturation

Heated at 65℃for 5min, rapidly quenched on ice and allowed to stand on ice for 2min.

(2) First strand cDNA Synthesis

And (5) centrifuging for a short time and mixing uniformly. Reacting at 55deg.C for 60min, heating at 70deg.C for 15min to terminate the reaction, and preserving the product at-20deg.C.

2.3 Gene amplification of interest

The glycosyltransferase genes AsUGT73E5 and AsUGT73E1 coding region primers AsUGT73E5-ORF-F/R, asUGT E1-ORF-F/R are designed. PCR amplification was performed using 5-fold diluted oat seedling cDNA as a template, and the target gene was PCR amplified using primers AsUGT73E5-ORF-F/R, asUGT E1-ORF-F/R and 2X Phanta Max Master Mix high-fidelity enzyme (vazyme, cat# P515-02), respectively, with the following reaction system:

reaction conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30sec, annealing at 30sec at a primer Tm value (AsUGT 73E5-ORF-F/R: 60℃and AsUGT73E1-ORF-F/R:60 ℃) and extension at 72℃for 1min for a total of 33 cycles; thoroughly extend at 72℃for 7min. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis.

As a result, as shown in FIG. 1, 1 was AsUGT73E5,2 was AsUGT73E1, and the gene bands were about 1500bp in size.

2.4 recovery of DNA gel

The target gene fragment was recovered using a Gel Extraction Kit (Omega, cat# D2500-02) kit, and the procedure was performed according to the kit instructions, as follows:

(1) Agarose gel containing the band of interest was cut out in an ultraviolet gum cutter, an equal volume of Binding Buffer was taken and the mixture incubated at 55℃for 7min until the gel was completely thawed.

(2) mu.L of the mixture was aspirated, transferred to a DNA adsorption column fitted with a 2mL collection tube, allowed to stand for 1min, centrifuged at 10,000g for 1min, and the filtrate was discarded.

(3) The adsorption column was placed in a recovery header, SPW Wash Buffer diluted with 700. Mu.L absolute ethanol was added, and the mixture was centrifuged at 10,000g for 1min, and the filtrate was discarded. Repeating once.

(4) The filtrate was discarded, the empty adsorbent column was placed back into the centrifuge tube and centrifuged at 12,000g for 2min.

(5) The empty adsorption column was placed in a sterilized 1.5mL centrifuge tube, the tube cap was opened and allowed to stand for 1min, 30. Mu.L of sterile water (preheated at 60 ℃) was added to the center of the adsorption membrane, and the mixture was allowed to stand at room temperature for 1min. The DNA was eluted by centrifugation at 12,000g for 1min.

2.5 cloning vector ligation

The gene of interest was cloned into pClone007 Blunt Simple Vector (Beijing engine family, cat# TSV-007 BS) and the reaction system was as follows:

reacting for 5min at room temperature.

2.6 E.coli transformation

(1) Taking 100 mu L of competent cells DH5 alpha (Shanghai Weidi organism) melted in an ice bath, adding target DNA, gently mixing, and standing in the ice bath for 30min;

(2) Heat shock is carried out for 60s in a water bath at the temperature of 42 ℃, and the centrifuge tube is rapidly transferred into an ice bath for 2min;

(3) 200 mu L of non-resistant sterile LB culture solution is added into the centrifuge tube, and after uniform mixing, the mixture is cultured for 1h at 180rpm in a shaking table at 37 ℃ to revive bacteria;

(4) Sucking the competent cells transformed in the previous step, adding the competent cells to LB agar medium containing ampicillin (Amp, screening concentration 100 mg/L) resistance, uniformly spreading the cells, drying the surface liquid of the medium, and culturing the flat plate at 37 ℃ overnight;

(5) Several single colonies are selected, added into 500 mu L LB liquid medium containing Amp resistance (100 mg/L), cultured for 4 hours at 37 ℃ and 180rpm, bacterial liquid PCR is identified, the identified primer is M13-F/R, positive clones are sent to Boston biotechnology limited company for sequencing, and cloning vectors pClone007-AsUGT73E5 and pClone007-AsUGT73E1 with correct sequences are obtained.

2.7 plasmid extraction

After bacterial protection of the correctly sequenced samples, plasmids were extracted using E.Z.N.A.plasmid Mini Kit I Kit (omega, cat# D6942-02) according to the instructions for use, using the following procedure:

(1) Taking 5mL of bacterial liquid (12-16 h) cultured overnight at 37 ℃, centrifuging at 10,000g for 1min, and discarding the supernatant;

(2) Adding 250 mu L of Solution I (RNase A is added) into the centrifuge tube, and blowing and uniformly mixing;

(3) Adding 250 μL Solution II, reversing for 4-6 times, mixing, standing for 2min to crack thallus (total time is less than 5 min);

(4) 350. Mu.L of Solution III was added and immediately turned upside down 6-8 times to allow the Solution to thoroughly mix, at which time a large amount of white flocculent precipitate appeared. Centrifuging at 13,000g for 10min;

(5) Placing the adsorption column in a collecting pipe, adding the supernatant after the suction and centrifugation into the adsorption column, centrifuging at 10,000g for 1min, and discarding the filtrate;

(6) 700. Mu.L of DNA Wash Buffer was added to the column, centrifuged at 10,000g for 1min, and the filtrate was discarded. Repeating the process once;

(7) Placing the empty adsorption column into a collecting pipe, centrifuging at 13,000g for 2min, transferring the adsorption column into a new 1.5mL centrifuge tube, opening a pipe cover to dry the adsorption column for 1min, and volatilizing the residual rinse liquid in the adsorption column;

(8) 50. Mu.L of sterile water preheated to 55℃was added to the center of the membrane of the adsorption column, left to stand for 2min and centrifuged at 13,000g for 1min. The adsorption column was discarded and the plasmid was stored at-20℃until use.

3. Protein expression

3.1 construction of prokaryotic expression vectors

PCR amplification was performed using pClone007 vector (pClone 007-AsUGT73E5 and pClone007-AsUGT73E 1) containing the target genes AsUGT73E5 and AsUGT73E1 open reading frame as templates, and recombinant primers AsUGT73E5-pGEXF/R, asUGT73E1-pGEXF/R were designed, and the reaction system and reaction conditions were the same as those described above 2.3, to obtain gene fragments for constructing expression vectors.

pGEX-6p-1 was used as a prokaryotic expression vector. pGEX-6p-1 vector was linearized with EcoRI (Thermo, cat# FD 0274) and SalI (Thermo, cat# FD 0644) endonucleases in the following system:

the reaction was terminated after 1 hour at 37 ℃.

PCR amplification products and linearized pGEX-6p-1 vector were recovered by agarose gel electrophoresis. The recovered gene fragment and linearized pGEX-6p-1 were subjected to homologous recombination according to the instructions using the ClonExpII One Step Cloning Kit kit (vacyme, cat# C112-02), and the ORFs of the genes AsUGT73E5, asUGT73E1 were cloned into the prokaryotic expression vector pGEX-6p-1, respectively, as follows:

the reaction is carried out for 30min at 37 ℃, the temperature is reduced to 4 ℃ or the reaction product is cooled on ice, and the obtained reaction product is transformed into escherichia coli DH5 alpha, and the transformation method is the same as 2.6. After overnight incubation, single colonies were picked, added to 500. Mu.L LB liquid medium containing Amp resistance (100 mg/L), incubated at 37℃for 4h at 180rpm, and identified by PCR in bacterial liquid, the identified primers were pGEX-F/R, and positive clones were sent to Bo XingKe Biotechnology Co., ltd for sequencing. The plasmid pGEX-AsUGT73E5/pGEX-AsUGT73E1 is extracted after the sample with correct sequencing result is preserved, and the plasmid extraction method is the same as the 2.7. Coli Rossetta (DE 3) expression competence (Shanghai-only organism) was transformed with the extracted plasmid, and pGEX-6p-1 empty vector transformation was set as a control, and the transformation method was the same as above 2.6.

3.2 protein-induced expression

The Rossetta (DE 3) bacterial solutions containing pGEX-AsUGT73E5/pGEX-AsUGT73E1/pGEX-6p-1 empty vector are respectively inoculated into 1L of LB liquid medium containing Amp resistance (100 mg/L) according to the volume ratio of 1:100, and cultured to OD by a shaking table at 37 ℃ at 200rpm ₆₀₀ =0.6, 0.2mm iptg was added and induced overnight at 16 ℃ with shaker 160 rpm. 4The cells were collected by centrifugation at 4,000rpm and 10mL of a pre-chilled PBS solution (0.01M, pH 7.4, formulation: naCl 8.0g, KCl 0.2g, na) ₂ HPO ₄ 1.44g，KH ₂ PO ₄ 0.24g, distilled water was added to 1L) and resuspended and sonicated on ice until the solution was translucent. Centrifugation was carried out at 12,000rpm for 15min at 4℃and the supernatant and pellet were collected and detected by SDS-PAGE.

3.3 protein purification

Equilibration/wash (equilibration and wash were formulated as well) and eluents were formulated and 1mM DTT was added prior to use. Balancing/washing solution (1L): 140mM NaCl,2.7mM KCl,10mM Na ₂ HPO ₄ ，1.8mM KH ₂ PO ₄ pH 7.4. Eluent (1L): 50mM Tris-HCl,10mM reduced glutathione, pH 8.0.

(1) Glutathione Beads (Hengzhou Tiandi people and organisms, goods number: SA 008010) is put into a proper chromatographic column, and is balanced by a balancing solution with the volume of 5 times of the column volume, so that the filler is under the same buffer system as the target protein, and plays a role in protecting the protein;

(2) Adding the sample into balanced Glutathione Beads, ensuring that the target protein is fully contacted with Glutathione Beads, improving the recovery rate of the target protein, and collecting effluent;

(3) Washing with a 10-time column volume of impurity washing liquid to remove nonspecifically adsorbed impurity proteins, and collecting the impurity washing liquid;

(4) Collecting eluent by using eluent with the volume of 5 times of the column volume, namely the target protein component;

(5) Sequentially using balance liquid with the volume of 3 times of column and deionized water with the volume of 5 times of column to balance filling;

(6) The purified protein solution was added to a millipore 15mL ultrafilter tube (10 KD), the sample was concentrated to 500. Mu.L by centrifugation at 4,000rpm, and 15mL PBS phosphate buffer (0.01M, formulation: naCl 8.0g, KCl 0.2g, na) was added ₂ HPO ₄ 1.44g，KH ₂ PO ₄ 0.24g, pH 7.4 was adjusted, distilled water was added to 1L, and concentration was continued to 500. Mu.L. Repeating the process once;

(7) Purified protein was aspirated, diluted and glycerol was added to a final concentration of 10% and stored at-80 ℃.

SDS-PAGE detects purified proteins. As a result, as shown in FIG. 2, 1 is an uninduced whole cell protein; 2 is whole cell protein after IPTG induction; 3 is the cell supernatant after IPTG induction; 4 is cell precipitation after IPTG induction; 5 is a protein purified by a supernatant GST column; and 6, purifying protein after ultrafiltration concentration. The recombinant proteins all appeared to have a distinct band around 80kDa compared to the uninduced control samples. The AsUGT73E5 and AsUGT73E1 proteins respectively comprise 515 and 490 amino acids, and the molecular weights of the fused AsUGT73E5 and AsUGT73E1 proteins are 82.21KDa and 79.79KDa respectively.

Example 2 functional identification of glycosyltransferases AsUGT73E5 and AsUGT73E1

1. Enzyme Activity assay

(1) AsUGT73E5 catalyzed glycosylation reaction

1mM steroidal sapogenin (diosgenin/pennogenin/neoacteoside), 1mM UDP-Glucose (UDP-Glc), 50. Mu.L of purified glycosyltransferase AsUGT73E5 was accurately weighed, and dissolved in PBS phosphate buffer (concentration 0.01M, formulation: naCl 8.0g, KCl 0.2g, na) ₂ HPO ₄ 1.44g，KH ₂ PO ₄ 0.24g, pH 8.0 was adjusted, distilled water was added to 1L, and the final volume was brought to 300. Mu.L. After 2h of reaction at 37 ℃, adding an equal volume of methanol to stop enzyme activity, and dissolving the product in 500 mu L of chromatographic methanol to be detected after the product is dried under reduced pressure.

(2) AsUGT73E1 catalyzed glycosylation reaction

The glycosylation reaction in (1) was repeated, and the whole was concentrated and dried to use as a substrate, 1mM UDP-Rhamnose (UDP-Rha) was added, 50. Mu.L of purified glycosyltransferase AsUGT73E1 was dissolved in PBS phosphate buffer (pH 8.0) to give a final volume of 300. Mu.L. After 2h of reaction at 37 ℃, adding an equal volume of methanol to stop enzyme activity, and dissolving the product in 500 mu L of chromatographic methanol to be detected after the product is dried under reduced pressure.

HPLC and LC-Q-TOF identification of enzyme products

(1) Liquid chromatography

The experiment used a Thermo UltiMate 3000 liquid chromatograph, thermo Hypersil GOLD C ₁₈ HPLC detection was performed on a liquid chromatography column (250 mm. Times.4.6 mm,5 μm). The mobile phases were water (a) and acetonitrile (B). ElutionGradient: 0-6 min, 20-30% B; 6-15 min, 30-60% B; 15-21 min, 60-100% B; 21-30 min,100% B; 30-35 min, 100-20% B. The flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and the detection wavelength is as follows: 210nm.

(2) Mass spectrometry detection

The experiment was performed using a AB SCIEX TripleTOF 6600 ultra-high resolution mass spectrometer. Positive ion data acquisition mode, the condition is: capillary voltage 3.6kV, taper hole voltage 35kV, ion source temperature 105 ℃, desolventizing gas temperature 340 ℃, reverse taper hole airflow 55L/h, desolventizing gas 650L/h, extraction taper hole 4V. Mass-to-charge ratio data scan range: 50-1500m/z.

Results and analysis

The product of reaction of AsUGT73E5 protein and diosgenin, UDP-Glc is dried by a vacuum concentrator, added with 500 mu L of chromatographic methanol for dissolution, filtered by a 0.22 mu m filter membrane and detected by HPLC. As shown in FIG. 3, the product of AsUGT73E5 showed a new peak (product 1) at 22.3min compared to the empty reaction, which was the same as the peak time of the trillin standard. The sample was recovered, and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a new product peak (product 2) was observed at 20.7min, which time was consistent with the rhizoma paridis saponin V standard. TOF positive ion scanning mode detection is shown in FIG. 4, the molecular weights of product 1 and product 2 are 577.38 (M+H) ⁺ ) And 723.43 (M+H) ⁺ ) The molecular weight of the Chinese medicinal materials is consistent with that of the trilobatin (576.3) and the paris polyphylla saponin V (722.4), which shows that the paris polyphylla saponin V is generated after the continuous catalysis of the AsiGT 73E5 and the AsiGT 73E1 by the diosgenin.

After HPLC detection of the product of the reaction of AsUGT73E5 protein with pennogenin, UDP-Glc (FIG. 5), the product of AsUGT73E5 showed a new peak at 18.8min (product 3) compared with the empty reaction. The sample was recovered, and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a new product peak (product 4) was observed at 17.1min, which time was consistent with the Paris polyphylla saponin VI standard. TOF positive ion scanning mode detection is shown in FIG. 6, the molecular weights of product 3 and product 4 are 593.37 (M+H) ⁺ ) And 739.43 (M+H) ⁺ ) With pennogenin-3-O-glucoside (592.3) and weightThe molecular weight of the paris saponin VI (738.4) is consistent, which shows that the paris saponin VI is generated after the continuous catalysis of the AsUGT73E5 and the AsUGT73E1 by the pennogen. The process of the AsUGT73E5 and AsUGT73E1 proteins catalyzing the glycosylation of steroidal sapogenins is shown in figure 7.

The product of reaction of AsUGT73E5 protein with neoacteosin and UDP-Glc is dried by a vacuum concentrator, dissolved by adding 500 mu L of chromatographic methanol, and filtered by a 0.22 mu m filter membrane for HPLC detection. As shown in FIG. 8, the product of AsUGT73E5 showed a new peak (product 5) at 16.7min, compared to the empty reaction. The sample was recovered and the reaction was continued by adding AsUGT73E1 protein and UDP-Rha, and a second product peak (product 6) was present at 15.6 min. TOF positive ion scanning mode detection is shown in FIG. 9, the molecular weights of product 5 and product 6 are 593.37 (M+H) ⁺ ) And 739.43 (M+H) ⁺ ) Indicating that the neotame sapogenin generates corresponding glycosylation products after being continuously catalyzed by AsUGT73E5 and AsUGT73E1. Reference is made to:

Cárdenas,P.D.,Sonawane,P.D.,Heinig,U.,Bocobza,S.E.,Burdman,S.,&Aharoni,A.(2015). The bitter side of the nightshades:Genomics drives discovery in Solanaceae steroidal alkaloid metabolism.Phytochemistry,113,24-32.

Christ,B.,Xu,C.C.,Xu,M.L.,Li,F.S.,Wada,N.,Mitchell,A.J.,…&Weng,J.K.(2019). Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.Nature Communications,10,3206.

Deng,D.,Lauren,D.R.,Cooney,J.M.,Jensen,D.J.,Wurms,K.V.,Upritchard,J.E.,…&Li,M.Z. (2008).Antifungal saponins from Paris polyphylla Smith.Planta Medica,74(11),1397-1402.

Gong,G.H.,Qin,Y.,Huang,W.,Zhou,S.,Wu,X.H.,Yang,X.H,…&Li.D.(2010).Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis.Chemico-Biological Interactions,184(3), 366-375.Journal of agricultural and food chemistry,64(7),1549-1556.

He,J.L.,Yu,S.,Guo,C.J.,Tan,L.,Song,X.M.,Wang,M.,…&Peng,C.(2019).Polyphyllin I induces autophagy and cell cycle arrest via inhibiting PDK1/Akt/mTOR signal and downregulating cyclin B1 in human gastric carcinoma HGC-27 cells.Biomedicine&Pharmacotherapy,117,109189.

Louveau,T.,Orme,A.,Pfalzgraf,H.,Stephenson,M.J.,Melton,R.,Saalbach,G.,...&Langdon,T. (2018).Analysis of two new arabinosyltransferases belonging to the carbohydrate-active enzyme(CAZY)glycosyl transferase family1 provides insights into disease resistance and sugar donor specificity.The Plant Cell,30(12),3038-3057.

Orme,A.,Louveau,T.,Stephenson,M.J.,Appelhagen,I.,Melton,R.,Cheema,J.,…&Osbourn, A.(2019).A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat.Proceedings of the National Academy of Sciences, 201914652.

Papadopoulou,K.,Melton,R.E.,Leggett,M.,Daniels,M.J.,&Osbourn,A.E.(1999). Compromised disease resistance in saponin-deficient plants.Proceedings of the National Academy of Sciences,96(22),12923-12928.

Qin,X.J.,Sun,D.J.,Ni,W.,Chen,C.X.,Hua,Y.,He,L.,&Liu,H.Y.(2012).Steroidal saponins with antimicrobial activity from stems and leaves of Paris polyphylla var.yunnanensis.Steroids,77(12),1242-1248.

Teng,J.F.,Qin,D.L.,Mei,Q.B.,Qiu,W.Q.,Pan,R.,Xiong,R.,…&Wu.,A.G.(2019). Polyphyllin VI,a saponin from Trillium tschonoskii Maxim.induces apoptotic and autophagic cell death via the ROS triggered mTOR signaling pathway in non-small cell lung cancer. Pharmacological Research,147,104396.

Vincken,J.P.,Heng,L.,de Groot,A.,&Gruppen,H.(2007).Saponins,classification and occurrence in the plant kingdom.Phytochemistry,68(3),275-297.

Wang,G.X.,Han,J.,Zhao,L.W.,Jiang,D.X.,Liu,Y.T.,&Liu,X.L.(2010).Anthelmintic activity of steroidal saponins from Paris Polyphylla.Phytomedicine,17(14),1102-1105.

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sequence listing

<110> university of northeast forestry

<120> oat glycosyltransferase AsUGT73E1 and its use in steroid saponin synthesis

<130> P200850-DBL

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 490

<212> PRT

<213> oat (Avena sativa l.)

<400> 1

Met Val Ala Ser Arg Val Lys Lys Leu Arg Val Leu Leu Ile Pro Phe

1 5 10 15

Phe Ala Thr Ser His Ile Glu Pro Tyr Thr Glu Leu Ala Ile Arg Leu

20 25 30

Ala Gly Ala Lys Pro Asp Tyr Ala Val Glu Pro Thr Ile Ala Val Thr

35 40 45

Pro Ala Asn Val Pro Ile Val Gln Ser Leu Leu Glu Arg Arg Gly Gln

50 55 60

Gln Gly Arg Ile Lys Ile Ala Thr Tyr Pro Phe Pro Ala Val Glu Gly

65 70 75 80

Leu Pro Ala Gly Val Glu Asn Leu Gly Lys Val Ala Ala Ala Asp Ala

85 90 95

Trp Arg Ile Asp Ala Ala Ala Ile Ser Asp Thr Leu Met Arg Pro Ala

100 105 110

Gln Glu Ala Leu Val Arg Ala Gln Ser Pro Asp Ala Met Val Ala Asp

115 120 125

Pro His Phe Ser Trp Gln Ala Gly Ile Ala Ala Asp Leu Gly Val Pro

130 135 140

Leu Val Ser Phe Ser Val Val Gly Ala Phe Ser Gly Leu Val Met Gly

145 150 155 160

Lys Leu Met Ala Tyr Gly Ala Val Glu Asp Gly Glu Asp Ala Val Thr

165 170 175

Ile Pro Gln Phe Pro Leu Pro Glu Ile Arg Ile Pro Val Thr Glu Leu

180 185 190

Pro Glu Phe Leu Arg Thr His Leu Leu Glu Arg Asp Gly Lys Asp Val

195 200 205

Asp Ser Ile Gly Lys Val Ser Val Gly Gln Asn Phe Gly Leu Ala Ile

210 215 220

Asn Thr Ala Ser His Leu Glu Gln Gln Tyr Cys Glu Met His Thr Ser

225 230 235 240

Gly Gly Gln Ile Lys Arg Ala Tyr Phe Val Gly Pro Leu Ser Leu Gly

245 250 255

Ala Glu Ala Val Ala Pro Gly Gly Gly Gly Gly Glu Thr Gln Ala Pro

260 265 270

Pro Cys Ile Arg Trp Leu Asp Ser Lys Pro Asp Arg Ser Val Val Tyr

275 280 285

Leu Cys Phe Gly Ser Leu Thr His Val Ser Asp Ala Gln Leu Asp Glu

290 295 300

Leu Ala Leu Gly Leu Glu Ala Ser Gly Lys Ala Phe Leu Trp Val Val

305 310 315 320

Arg Ala Ala Glu Ala Trp Arg Pro Pro Ala Gly Trp Ala Glu Arg Val

325 330 335

Gln Asp Arg Gly Met Leu Leu Thr Ala Trp Ala Pro Gln Thr Ala Ile

340 345 350

Leu Gly His Arg Ala Val Gly Ala Phe Val Thr His Cys Gly Trp Asn

355 360 365

Ser Val Leu Glu Ala Val Ala Ala Gly Leu Pro Val Leu Thr Trp Pro

370 375 380

Met Val Phe Glu Gln Phe Ile Thr Glu Arg Leu Val Thr Glu Val Met

385 390 395 400

Gly Ile Gly Glu Arg Phe Trp Pro Glu Gly Ala Gly Arg Arg Ser Thr

405 410 415

Arg Tyr Glu Glu His Gly Leu Val Pro Ala Glu Asp Val Ala Arg Ala

420 425 430

Val Thr Thr Phe Met Cys Pro Gly Gly Ala Gly Asp Ala Lys Arg Gln

435 440 445

Arg Ala Met Glu Leu Ala Ala Glu Ser Arg Ala Ala Met Ala Glu Gly

450 455 460

Gly Ser Ser His Arg Asp Leu Cys Arg Leu Val Asp Asp Leu Val Ala

465 470 475 480

Ala Lys Leu Glu Arg Glu Gln Val Pro Ser

485 490

<210> 2

<211> 1473

<212> DNA

<213> oat (Avena sativa l.)

<400> 2

atggttgcca gccgtgtgaa gaagctgcgt gtcctgctca ttcccttctt cgcgacaagc 60

cacatcgagc cctacaccga gctcgccatc cgcctcgccg gcgccaagcc ggactacgcc 120

gtggagccaa caattgcggt gacgccggcg aacgtcccaa tcgtccagtc cttgctggag 180

cgacgcggac agcaggggcg catcaagatc gcgacgtacc cgttcccggc cgtggagggc 240

ctcccggcgg gcgtggagaa cctgggcaag gtcgcggcgg ccgacgcctg gcgcatcgac 300

gcggccgcca tcagcgacac cctgatgcgg cccgcgcagg aggcgctggt gagggcgcag 360

tcccccgacg ccatggtcgc cgacccgcac ttctcctggc aggccggcat cgccgccgat 420

ctgggcgtgc cgctggtgtc gttcagcgtg gtgggcgcct tctcggggct cgtcatgggc 480

aaactcatgg cctacggcgc cgtcgaggac ggcgaagacg ccgttacgat ccctcagttt 540

ccccttccgg agatacggat accggtgacc gagctgccgg agttcctgag gacccacctg 600

ctcgagcgtg acgggaagga cgtcgatagc atcggcaaag tttcggtggg acagaatttc 660

ggcctcgcca tcaacacggc gtcgcacctg gagcagcagt actgcgagat gcacaccagc 720

ggcggccaaa tcaagcgagc ctacttcgtg gggcccctct cgctgggagc cgaagcagtt 780

gcccccggcg gcggcggcgg cgagacacag gcgccgccgt gcatccgttg gctggactcg 840

aagccggacc ggtcggtggt gtacctgtgc ttcgggagcc tgacccacgt ctcggacgcg 900

cagctggacg agctggctct cgggctggag gcgtccggga aggcgttcct gtgggtggtg 960

agggcggcgg aggcgtggcg gccgccggcg gggtgggcgg agcgcgtgca ggacaggggg 1020

atgctcctga ccgcctgggc cccgcagacc gccatcctgg gccaccgcgc cgtgggcgcc 1080

ttcgtgacgc actgcgggtg gaactcggtg ctggaggcgg tggcggcggg gctgccggtg 1140

ctgacgtggc cgatggtgtt cgagcagttc atcacggaga ggctggtgac ggaggtgatg 1200

gggatcgggg agcggttctg gccggagggc gccggacggc ggagcaccag gtacgaagag 1260

cacgggctgg tcccggcgga ggacgtggcg cgggcggtga caacgttcat gtgccccgga 1320

ggagcagggg acgccaagag gcagagggcg atggagctcg ccgccgagtc tcgtgcggcc 1380

atggcggaag gaggctcgtc gcaccgtgat ctgtgccgcc tcgttgacga tctcgtcgca 1440

gctaagctag agagagagca ggtgcctagc tag 1473

<210> 3

<211> 515

<212> PRT

<213> oat (Avena sativa l.)

<400> 3

Met Ala Asp Leu His Phe Leu Val Val Pro Leu Ala Ala Gln Gly His

1 5 10 15

Ile Ile Pro Met Val Asp Val Ala Arg Leu Leu Ala Ala Arg Gly Ser

20 25 30

Arg Val Thr Val Val Thr Thr Pro Val Asn Ala Ala Arg Asn Arg Ala

35 40 45

Ala Val Asp Gly Ala Arg Lys Ala Gly Leu Ala Val Glu Leu Leu Glu

50 55 60

Leu Pro Phe Pro Ser Ala Gln Leu Gly Leu Pro Glu Gly Leu Glu Ala

65 70 75 80

Val Asp Gln Leu Asn Gly Gln Pro Pro Glu Ile Ser Ile Gly Leu Phe

85 90 95

Lys Ala Ile Trp Thr Leu Ala Gly Pro Leu Glu Glu Tyr Leu Arg Ala

100 105 110

Leu Pro Arg Leu Pro Asp Cys Leu Val Ala Asp Leu Cys Asn Pro Trp

115 120 125

Thr Ala Pro Val Cys Glu Arg Leu Gly Ile Pro Arg Leu Val Met His

130 135 140

Cys Pro Ser Ala Tyr Phe Gln Leu Ala Val His Arg Leu Asn Glu His

145 150 155 160

Gly Val Tyr Gly Gly Gly Val Glu Asp Tyr Asp Pro Thr Pro Ile Glu

165 170 175

Val Pro Gly Phe Pro Val Arg Ala Phe Gly Ser Lys Thr Thr Met Arg

180 185 190

Gly Phe Phe Gln Tyr Pro Gly Val Glu Gln Glu His Leu Glu Ala Leu

195 200 205

His Ala Glu Ala Thr Ala Asp Gly Leu Leu Phe Asn Ser Phe Arg Ala

210 215 220

Ile Glu Ala Asp Phe Leu Asp Ala Tyr Ala Ala Ala Leu Gly Lys Thr

225 230 235 240

Thr Trp Ala Val Gly Pro Thr Ala Leu Val Asn Asp Thr Thr Thr Thr

245 250 255

Thr Ala Ser Ser Arg Ser Ser Thr Ile Val Ser Trp Leu Asp Ala Arg

260 265 270

Pro Pro Asp Ser Val Leu Tyr Val Ser Phe Gly Ser Ile Ser Leu Leu

275 280 285

Ser Ala Lys Gln Leu Ala Lys Leu Ala Asp Gly Leu Glu Ala Ser Gly

290 295 300

Arg Pro Phe Val Trp Ala Ile Lys Glu Asp Lys Ala Asp Ala Ala Val

305 310 315 320

Arg Ser Gln Leu Asp Glu Glu Gly Gly Phe Glu Ala Arg Val Lys Asp

325 330 335

Arg Gly Leu Leu Val Arg Gly Trp Ala Pro Gln Val Ala Ile Leu Ser

340 345 350

His Pro Ala Val Gly Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr

355 360 365

Leu Glu Ala Leu Ser His Gly Val Pro Ala Leu Thr Trp Pro Thr Asn

370 375 380

Ala Asp Gln Phe Cys Ser Glu Gln Val Ile Val Asp Val Leu Asp Val

385 390 395 400

Gly Val Arg Ser Gly Val Lys Ile Pro Ala Leu Tyr Val Pro Pro Glu

405 410 415

Ala Glu Gly Val Gln Val Glu Ser Gly Asp Val Glu Arg Ala Ile Val

420 425 430

Glu Leu Met Asp Gly Gly Pro Glu Gly Ala Ala Arg Arg Ala Arg Ala

435 440 445

Arg Lys Ile Ala Val Glu Ala Lys Ala Ala Met Glu Glu Gly Gly Thr

450 455 460

Ser His Ser Asp Leu Thr Asp Met Ile Arg His Val Ser Glu Leu Ser

465 470 475 480

Arg Lys Lys Arg Leu Gln Leu Glu Thr Ala Asp Ala Thr Cys Glu Glu

485 490 495

Ala Thr Arg Ala Ala Asp Asn Ala Ala Ala Val Leu Pro Leu Leu Ser

500 505 510

Gln Ala Asn

515

<210> 4

<211> 1548

<212> DNA

<213> oat (Avena sativa l.)

<400> 4

atggcggatc tacacttcct ggtcgtgccg ctggcggcgc agggccacat catccccatg 60

gtggacgtgg cgcgcctcct cgccgcgcgt ggctcgcggg tcaccgtcgt caccacgccc 120

gtcaacgccg cgcgcaaccg ggccgccgtg gacggcgcca ggaaggcggg cctcgccgtc 180

gagctcctgg agctcccgtt ccccagcgcg cagctcggcc tgcctgaggg cctggaggcc 240

gtcgaccagc tgaacgggca gccacctgaa atctccatcg gcctcttcaa ggccatctgg 300

accctggccg gaccgctgga ggagtacctc cgcgcgctgc cgcgcctgcc ggactgcctc 360

gtcgccgact tgtgcaaccc ttggacggcg ccggtctgcg agcgcctcgg catcccgagg 420

ctggtgatgc actgcccgtc cgcctacttc cagctcgccg tgcaccgcct gaacgagcac 480

ggcgtgtacg gcggaggcgt cgaggactac gaccccacgc ctatcgaggt gccgggcttc 540

cccgtgcgcg ccttcgggag caagaccacc atgcggggct tcttccagta ccccggcgtc 600

gagcaggagc accttgaagc gctccacgcc gaggccaccg ccgacggcct gctcttcaac 660

agcttccgcg ccatcgaggc cgacttcctc gacgcctacg cggcggcgct cggcaagacg 720

acgtgggccg tcgggccgac cgccttggtg aacgacacca ccaccaccac cgcctcctcg 780

aggtcgagca ccatcgtgtc gtggctcgac gcccggccgc cggactccgt gctgtacgtc 840

agcttcggca gcatctccct gctgtcggcg aagcagctgg cgaagctcgc ggacgggctg 900

gaggcgtcgg ggcggccgtt cgtgtgggcg atcaaggagg acaaggcgga cgcggcggtg 960

cggtcgcagc tggacgagga gggagggttc gaggcgcggg tcaaggacag gggcctcctg 1020

gtgcgcgggt gggcgccgca ggtggccatc ctctcgcacc cggcggtggg cggcttcctc 1080

acgcactgcg gctggaacag cacgctggag gccctctcac acggcgtgcc ggcgctgacg 1140

tggcccacca acgccgacca gttctgcagc gagcaggtga tcgtggacgt cctcgacgtc 1200

ggcgtcaggt ctggcgtcaa gatcccggcc ctgtacgtgc ccccggaggc cgagggggtg 1260

caggtggaga gcggcgacgt ggagagggcg atcgtggagc tgatggacgg cgggccggag 1320

ggagcggcga ggagggccag ggcaaggaag attgccgtgg aggccaaggc ggccatggag 1380

gaaggcggga cgtcgcactc cgacctaacg gacatgatcc gccatgtctc ggagctgtcc 1440

aggaagaaga ggctccagct cgagacagcc gacgcgacct gtgaagaagc aacaagagca 1500

gcagacaacg ctgccgcagt actgcctcta ctgtcccaag ctaattaa 1548

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggcggatc tacacttcct 20

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ttaattagct tgggacagta ga 22

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atggttgcca gccgtgtga 19

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ctagctaggc acctgctct 19

<210> 9

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gcccctggga tccccggaat tcatggcgga tctacacttc ct 42

<210> 10

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cgatgcggcc gctcgagtcg acttaattag cttgggacag taga 44

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gcccctggga tccccggaat tcatggttgc cagccgtgtg a 41

<210> 12

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cgatgcggcc gctcgagtcg acctagctag gcacctgctc t 41

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tgtaaaacga cggccagt 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

caggaaacag ctatgacc 18

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cagcaagtat atagcatggc c 21

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ggagctgcat gtgtcagagg 20

Claims

1.A glycosyltransferase has an amino acid sequence shown in SEQ ID NO. 1.

2.A gene encoding the glycosyltransferase of claim 1.

3. The gene according to claim 2, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

4. An expression cassette, vector or recombinant bacterium comprising the gene of claim 2 or 3.

5. The method for producing glycosyltransferase of claim 1, comprising the steps of: constructing the expression vector of the gene as claimed in claim 2 or 3, introducing the expression vector into an expression host bacterium to obtain recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, collecting the thallus, and extracting and purifying the protein.

6. Use of the glycosyltransferase of claim 1 for the synthesis of steroid saponins, characterized in that: in the synthesis of the steroid saponin, the substrate is the trillion grass glycoside or the pennogenin-3-O-glucoside, and the rhamnosyl is introduced under the action of glycosyltransferase to generate the paris saponin V or the paris saponin VI.

7.A method for synthesizing paris polyphylla saponin V comprises the following steps: the glycosyltransferase of claim 1 is used to react with trillin and UDP-Rha to generate parietal saponin V.

8.A method for synthesizing paris polyphylla saponin VI comprises the following steps: the glycosyltransferase of claim 1 is used to react with pennogenin-3-O-glucoside and UDP-Rha to generate paridis saponin VI.